The long-term goal of this research is to understand, at the molecular level, how transcription elongation is regulated in eukaryotic cells. The importance of appropriate regulation of transcription elongation in human cells has recently been highlighted by the discovery that the gene alterations associated with several forms of cancer alter the function of cellular transcription elongation factors. Research in this laboratory focuses on human and yeast RNA polymerase II, the enzyme that transcribes nuclear protein-encoding genes. The proposed project is a combined biochemical and genetic approach aimed at identifying the physiological role of a recently discovered nuclease activity intrinsic to this and other eukaryotic and prokaryotic RNA polymerases. The conservation of this activity suggests that it is functionally important for transcription. However, whether this activity is required for accuracy of RNA synthesis, efficient RNA chain elongation, transcription termination, and/or regulation of gene expression is not known for any RNA polymerase. Furthermore, mutants that lack this activity have not been described. The proposed investigation will use several strategies to isolate such mutants in the yeast Saccharomyces cerevisiae. Enzymes shown to have an impaired nuclease activity will be assayed in vitro to identify possible alterations in other properties, including the rate and fidelity of transcription elongation and the propensity to pause, arrest, or terminate transcription. The observed biochemical properties and physiological behavior of these mutant enzymes will serve as the basis for proposing and testing specific hypotheses addressing nuclease function in vivo and in vitro. In addition, the physiological role of the 511 protein, which stimulates the nuclease activity, will also be investigated with the proposed experiments. In particular, the re~arch will explore the possibility that the nuclease activity has several functions, perhaps only a subset of which are dependent on SII. Similarly, experiments are proposed to determine whether SII has an additional role in transcription, independent of its stimulation of the nuclease. Together, these experiments will provide insight into the importance of an activity that, until recently, was completely unknown but which is likely to be central to the accurate function and regulation of RNA polymerases.